Colonization of the human gastric mucosa by Helicobacter pylori is associated with an increased risk for development of peptic ulcer disease and distal gastric adenocarcinoma. Studies in a mouse model for H. pylori infection indicate that expression of a toxin (VacA) enhances the capacity of H. pylori to colonize the stomach, and immunization of mice with VacA results in protective immunity. VacA contributes to gastric mucosal damage, and analysis of vacA alleles in H. pylori isolates from humans suggests that VacA plays a role in the pathogenesis of peptic ulcer disease. The effects of VacA on eukaryotic cells include vacuolation, altered trafficking within the endocytic pathway, membrane channel formation, and apoptosis. The VacA mechanism of action remains incompletely understood. Based on our preliminary studies, we hypothesize that the mature VacA toxin can be divided into three functional domains: (i) an N-terminal hydrophobic region (amino acids 1-32) involved in membrane insertion, transmembrane protein dimerization, and membrane channel formation; (ii) an N-terminal region (amino acids 33-422) that is required for intracellular toxin activities (cell vacuolation and apoptosis); and (iii) a C-terminal domain (amino acids 423-821) involved in binding of VacA to eukaryotic cells. This proposal outlines plans for in-depth structure-function analysis of these three domains. We will use multiple experimental approaches, including several mutagenesis strategies, assays of VacA channel activity, an in vitro system for analyzing peptide insertion into membranes, expression of recombinant VacA, a system for intracellular VacA expression, mapping of VacA structure with recombinant anti-VacA antibodies, and use of a mouse model to examine the functions of VacA in vivo. These studies should result in a better understanding of VacA structure and function, and insights into the VacA mechanism of action. Ultimately, these studies may lead to advances in the treatment or prevention of H. pylori-associated human diseases.